3116 - High Environmental L-Lactate Facilitates DNA Damage Repair Pathway by Inducing Lactylation in Cervical Carcinoma Cells
Presenter(s)
D. Negrón-Figueroa1, R. Wang1, J. S. Gonzalez Diaz1, M. B. El Alam1, E. J. Lynn1, S. F. Ehsan1, and L. Colbert2; 1Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 2Division of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX
Purpose/Objective(s): We previously demonstrated that L-lactic acid producing bacteria, such as Lactobacillus iners, in the tumor microenvironment can lead to radiation resistance via enhanced DNA damage repair (DDR). Lactate can alter protein function by post-translational modifications (PTM), primarily lactylation of key lysines. Other studies have demonstrated that lactylation of key homologous recombination proteins, such as MRE11 and NBS1, can enhance DDR independently of other long-known PTM, such as acetylation. Lactylation of key histones also increases chromatin accessibility and facilitates damage repair. We hypothesize that these are potential mechanisms by which these lactate producing bacteria enhance DDR and cause radiation resistance. The purpose of this study was to quantify lactylation of key histones and DDR genes after lactate exposure.
Materials/Methods: We generated lactate-adapted HeLa cell lines with exposure to L-Lactic Acid (LA) and Sodium Lactate (NALA) in their media for 1-3months. First, we exposed the lactate-adapted pretreated HeLa cell lines to different doses of radiation (2.5Gy and 10Gy). Then, after radiation exposure, we collected lysates at different timepoints: 1hr, 2hrs, and 6hrs for the control and lactate-adapted HeLa cells. At last, we performed Western Blots of the resulting lactate-adapted cells.
Results: For the lactate-adapted HeLa cell lines, for most of LA and NALA treatment, there was an increase in lactylation, specially for proteins upstream of homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. We calculated the ratio of lactylation or phosphoralytion (intensity of protein with targeted PTM/intensity of total protein or tubulin) for LA and NALA pre-treatment. Then, we compared lactate-adapted groups to parental cells with and without radiation. Both LA and NALA could induce phosphorylation of DNA-pkcs (1.47 and 1.46, respectively) and key histones, H4K5 (1.87, 1.64), and for H4K8 (1.61, 1.27). After radiation, there was a further increase of lactylation of H3K18 (1.68) and H4K8 (1.38) in the LA-adapted cells, H4K5 (1.37) in the NALA-adapted cells. Radiation induced further phosphorylation of DNA-pkcs in both LA- and NALA-adapted cells (2.89, 3.72), and ATM (3.04) in the LA-adapted cells. We didn’t detect an increase of lactylation of MRE11 and NBS1 in the cells without radiation. After radiation, NALA induced MRE11 lactylation (1.14), while LA induced NBS1 lactylation (1.23).
Conclusion: We will perform more replicates and then analyze the data statistically. With this, future work will entail expanding this line of experiments to other cervical cancers cell lines. Furthermore, to investigate if lactylation level alteration could lead to variance response to radiation treatment, we would test the viability of the lactate-adapted cells vs parental cells when exposed to radiation.